Various publications are referred to in parentheses throughout this application. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
Breast cancer is the most prevalent malignant disease of women in the developed world, apart from non-melanoma skin cancers, with approximately one in eight women in the United States being diagnosed with breast cancer at some time in their lives. Breast cancer mortality is largely attributable to the development of systemic, hematogenous metastatic disease. Although approximately 10-15% of patients have an aggressive form of the disease that metastasizes within three years after initial diagnosis, the clinical manifestations of occult metastatic disease can appear ten or more years later. The consequence is that most breast cancer patients carry a risk for development of metastatic disease throughout the remainder of their natural lives (Ries et al., 2004; Weigelt et al., 2005).
To decrease the risk for the emergence of metastatic tumors, approximately 80% of breast cancer patients are treated with adjuvant chemotherapy. The clinical benefit is a 3 to 10% increase in 15-year survival, depending upon the age of the patient at diagnosis. However, since only about 40% of these patients eventually relapse and develop metastatic disease, there is a significant subset of patients who are unnecessarily subjected to the acute and long-term side effects of current chemotherapeutic regimens (Weigelt et al., 2005). Currently established clinical prognostic criteria such as the histopathological grade of the tumor or tumor size do not successfully predict systemic metastatic potential. Even angiolymphatic invasion and the presence of regional lymph node metastases do not always correlate with subsequent distant spread. This may be because the mechanisms of hematogenous spread are different from those for lymphatic spread. The ability to identify at the time of diagnosis those tumors that have increased likelihood for systemic hematogenous spread would aid in tailoring therapeutic interventions specific for a particular patient and identify those patients who would benefit the most from systemic therapy.
The tumor microenvironment is critical for facilitating metastasis. Utilizing intravital multiphoton imaging in rat and mouse mammary tumors, invasive carcinoma cells have been shown to polarize, move toward, and invade blood vessels (Wyckoff et al., 2007). This polarization and increased motility, leading to invasion of blood vessels by carcinoma cells, requires a paracrine loop involving macrophage-derived epidermal growth factor (EGF) and carcinoma cell-derived colony stimulating factor-1 (CSF-1) (Wyckoff et al., 2004, 2007). Invasive carcinoma cells involved in this paracrine loop yield a distinct gene expression profile, called the invasion signature, and increased cell motility and invasion are the result of alterations in the expression of cytoskeletal regulatory proteins (Wang et al., 2004, 2007).
A key actin polymerization regulatory protein that is part of the invasion signature and that is up-regulated in invasive tumor cells is Mena (Goswami, et al., 2009; Wang et al., 2004, 2007), an Ena/VASP protein family member that is highly conserved across species (Gertler et al., 1996; Krause et al., 2003). Mena regulates cell movement by its ability to protect actin filaments from capping proteins during polymerization (Barzik et al., 2005). Mena is up-regulated in malignant human breast tumors (Di Modugno et al., 2004, 2006, 2007) and is over expressed in a subpopulation of invasive tumor cells of rat and mouse mammary tumors (Wang et al., 2004, 2007), suggesting that Mena plays a central role in regulating breast carcinoma cell invasion. The forced expression of Mena in tumor cells at levels observed in invasive tumor cells has shown that Mena promotes carcinoma cell motility and invasiveness both in vivo and in vitro, and increases intravasation and lung metastasis in vivo. Mena stabilizes invadopodia, actin-rich protrusions that contain proteases, thereby increasing the matrix degradation activity of tumor cells. Importantly, Mena activity potentiates EGF-induced carcinoma cell invasion and membrane protrusion. In aggregate these results indicate that the upregulation of Mena expression in invasive tumor cells enable them to invade and metastasize in response to otherwise benign EGF stimulus levels thereby being more responsive to macrophage signaling (Ulrike et al., 2008).
The density of tumor-associated macrophages (TAM) has been suggested to be a prognostic marker of poor outcome for a variety of carcinomas, including breast carcinoma (Bingle et al., 2002; Condeelis et al., 2006). Macrophages comprise a key component of the tumor microenvironment as facilitators of tumor cell migration and intravasation, stromal matrix breakdown, and angiogenesis (Condeelis et al., 2006). In murine mammary tumors resulting from the expression of the PyMT oncogene in wild-type mice with intact macrophage numbers and function, carcinoma cells, when associated with macrophages, demonstrate an invasive phenotype with increased motility (Wyckoff et al., 2004). Tumor cell motility occurs more than 80% of the time in association with macrophages. Extensive multiphoton time lapse imaging of live tumors has shown that tumor cell intravasation was only observed in association with perivascular macrophages and was not seen in regions of blood vessels without perivascular macrophages. A seven fold reduction in the number of perivascular macrophages in csf1op/csf1op mice was correlated with a 10 fold reduction of circulating tumor cells in the blood of the same mice. Therefore, while tumor cell intravasation in the absence of perivascular macrophages cannot be ruled out, it has not been detected in vivo by direct imaging and may be only a minor kinetic component of intravasation, while perivascular assisted tumor cell intravasation appears to be a major portal of entry of tumor cell into the blood (Wyckoff et al., 2007).
Thus, there is a need for reliable methodologies to predict the risk for metastatic disease in cancer patients in order both to administer proper treatment to patients whose tumors have a high risk of metastasizing and to avoid unnecessary administration of chemotherapy to patients whose tumor had a negligible risk of metastasizing. The present invention addresses this need.